Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and lysosomal storage disorders (LSD), affect a large population of patients. For example, PD has a prevalence of 1-2% in people over the age of 50, worldwide distribution, and no gender preference (Kordower and Aebischer, 2001). The common hallmark of neurodegenerative diseases is the selective loss of neurons in different brain regions and in the spinal cord (Djaldetti and Melamed, 2001). PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN), resulting in resting tremor, rigidity, bradykinesia, and postural imbalance (Goetz et al., 1989). Despite many years of focused research, the causes of this disease remain to be elucidated (Steece-Collier et al., 2002). Levodopa and other dopaminergic medications significantly improve the motor symptoms and quality of life of patients with PD in the early stages of the disease. L-Dopa is most successful during the first few years of treatment, and this period is known as the L-dopa honeymoon. However, once the honeymoon period has waned, patients become progressively more disabled, despite an ever more complex combination of available anti-Parkinsonian treatments (Goetz et al., 1989). Sooner or later, they suffer from dopa-resistant motor symptoms (speech impairment, abnormal posture, gait and balance problems), dopa-resistant non-motor signs (autonomic dysfunction, mood and cognitive impairment, sleep problems, pain) and/or drug-related side effects (especially psychosis, motor fluctuations, and dyskinesias) (Rascol et al., 2003). To fill this therapeutic gap, pallidotomy, deep-brain stimulation, and transplantation of fetal midbrain dopamine-producing neurons have been developed. Nevertheless, the current status of PD treatment cannot be considered as ideal with regard to either efficacy or safety (Djaldetti and Melamed, 2001; Du et al., 2001; Shastry, 2001), although it is favorable as compared with other neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis (Deglon and Aebischer, 2002).
Chronic granulomatous disease (CGD) is an inherited disorder of host defense, in which the generation of superoxide and derivative microbicidal oxidants by the NADPH oxidase in phagocytic leukocytes is absent or markedly deficient due to mutations in oxidase subunit gp91phox, p47phox, or others. Victims suffer from recurrent and often life-threatening bacterial and fungal infections beginning in early childhood. Chronic inflammatory granulomas, a hallmark of CGD, can obstruct internal organs such as ureter and bowel. Although daily administration of prophylactic antibiotics plus interferon-γ decreases the frequency of infection and allogeneic bone marrow transplantation from HLA-identical donors cures selected patients, the mortality rates are still 2-4% annually. Because CGD results from specific gene defects in hematopoietic stem cells (HSCs), and mouse models that recapitulate the human disease have been developed, CGD has become an attractive target disorder for gene therapy.
Atherosclerosis is the leading cause of mortality in developed countries, accounting for nearly 50% of all deaths. Common risk factors include high cholesterol, diabetes, hypertension, smoking, obesity, and a familial predisposition. Interventions targeting these factors have had limited effects. HMG CoA reductase inhibitors (statins), a potent class of cholesterol-lowering drugs, have been proven to reduce cardiovascular mortality in hypercholesterolemic patients. Percutaneous and surgical treatments are aimed at either disrupting or bypassing flow-limiting lesions. Stem cell and gene therapy holds great promise, but is in its infancy1.
Atherosclerosis is a disorder of lipid metabolism, as well as a chronic inflammatory disease. Macrophages, participating in both lipid metabolism and inflammation, have key roles in all phases of atherosclerosis, from development of the fatty streak to processes that ultimately contribute to plaque rupture and MI. Macrophage expression of a number of genes may protect against atherosclerosis and inadequate expression or lack of expression of these genes leads to atherogenesis. They are apoE, apoAI, ABCA1, HSL, LXR, and PPAPγ, among others. On the other hand, some genes expressed in macrophages are involved in atherogenesis, such as CCR2, MCP-1, CCR5, MCSF, COX-2, 12/15-LO, and macrophage fatty-acid-binding protein aP2. There are a number of genes that can be manipulated in macrophages to benefit patients with atherosclerosis. For instance, apoE, apoAI, ABCA1 (ATP-binding cassette transporter A1), and LXRs (liver X receptors) are among genes to be over-expressed, whereas CCR2, 12/15-lipoxygenase, and macrophage fatty-acid-binding protein aP2 are candidate genes to be knocked down by the powerful RNAi technique.
Many viral promoters, such as CMV, show strong promoter activity, but are generally non-selective, acting in a wide variety of cell types. Lacking cell specificity, they may drive inappropriate gene expression in non-target tissues and cells causing additional problems for the recipient. Furthermore, it was reported that the level of gp91phox expression resulting from transduction with a CMV-driven lentivector was probably inadequate for clinical application 36. On the other hand, native promoters are either too long to incorporate into the vector used or too weak to drive transgene expression effectively. In studies on roles of interactions between transcription factors and cis-elements in gene regulation, tandem repetitive cis-elements have been successfully used to amplify function. Repetitive regulatory elements are also engineered into other types of constructs. In the tetracycline-regulated system, expression of the gene of interest is controlled by a promoter that contains seven tetracycline response elements (TRE)37,37,38. Recently, synthetic muscle promoters have been developed with activity exceeding naturally occurring promoter sequences39. Random assembly of E-box, MEF-2, TEF-1, and SRE sites into synthetic promoter recombinant libraries led to the isolation of several artificial promoters whose transcriptional potencies greatly exceed those of natural myogenic and viral gene promoters39,40.
The present invention overcomes previous shortcomings in the treatment of various disorders such as the ones described above by providing methods and compositions whereby macrophages are engineered to selectively express therapeutic nucleic acids under the control of super macrophage promoters to deliver therapeutic peptides and proteins to the site of disease lesions.